Enzymatic process for the resolution of enantiomeric mixtures of beta-lactams

ABSTRACT

A process for the resolution of an enantiomeric mixture of β-lactams containing an ester, the process comprising selectively hydrolyzing the ester of one of the enantiomers by combining the mixture with homogenized liver.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/688,852 filed on Oct. 16, 2000, which claims the benefit ofU.S. Provisional Application No. 60/160,103 filed Oct. 18, 1999.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to enzymatic processes for theresolution of enantiomeric mixtures of β-lactams useful in thepreparation of taxanes.

[0003] The taxane family of terpenes, of which taxol and docetaxel aremembers, has attracted considerable interest in both the biological andchemical arts. Such taxanes may be prepared through a variety ofsemi-synthetic routes. In one, a β-lactam is coupled to a derivative of10-deacetylbaccatin III to form a sidechain at the C-13 position of thederivative. As the stereochemistry of these taxanes may affect theirpharmaceutical activity, methods allowing efficient stereospecificpreparation of the intermediate β-lactam, as well as the final taxaneproducts, have been the subject of investigation.

[0004] Brieva et al. (Brieva, R.; Crich, J. Z. and Sih, C. J., J. Org.Chem. 1993,58, 1068) reported that racemic β-lactam underwent selectivekinetic hydrolysis with several Pseudomonas lipases and twopenicillinases. Pseudomonas lipases used by Brieva et al. include P-30,AK and K-10.

[0005] Similarly, Patel reported in U.S. Pat. No. 5,879,929, thatenantiomeric mixtures of certain β-lactams and, in particular, racemicmixtures of certain β-lactams, can be resolved by a stereoselectivehydrolysis using a variety of lipases and enzymes. Lipases identified byPatel include Amano PS-30 (Pseudomonas cepacia), Amano GC-20 (Geotrichumcandidum), Amano APF (Aspergillus niger), Amano AK (Pseudomonas sp.),Pseudomonas fluorescens lipase (Biocatalyst Ltd.), Amano Lipase P-30(Pseudomonas sp.), Amano P (Pseudomonas fluorescens), Amano AY-30(Candida cylindracea), Amano N (Rhizopus niveus), Amano R (Penicilliumsp.), Amano FAP (Rhizopus oryzae), Amano AP-12 (Aspergillus niger),Amano MAP (Mucor meihei), Amano GC-4 (Geotrichum candidum), Sigma L-0382and L-3126 (porcine pancrease), Lipase OF (Sepracor), Esterase 30,000(Gist-Brocarde), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp.,Amano), Sigma L-3001 (Wheat germ), Sigma L-1754 (Candida cylindracea),Sigma L-0763 (Chromobacterium viscosum) and Amano K-30 (Aspergillusniger). Enzymes identified by Patel include enzymes derived from animaltissue such as esterase from pig liver, α-chymotrypsin and pancreatinfrom pancreas such as Porcine Pancreatic Lipase (Sigma). While theseenzymes may be used in the stereoselective hydrolysis of β-lactams, therequired purification of the enzyme can significantly increase the costof the preparation of the β-lactam.

[0006] Whitesell et al. (Whitesell, J. K. Lawrence, R. M. Chimia, 1986,40, 315) and Basavaiah et al. (Basavaiah, D. and Rao, P. Tetrahedron.Asym., 1994, 5, 223-234) reported successful application of pig liveracetone powder (PLAP), bovine liver acetone powder (BLAP) and chickenliver acetone powder (CLAP), in the resolution of numerous chiralsecondary alcohols. Experimental evidence obtained to date, however,suggests that the use of these materials results in a product havingrelatively low optical purity. While the reason for this is not entirelyclear, it is believed that this is the result of incomplete reactionrather than the enzyme's lack of selectivity which, in turn, is likely aconsequence of inconsistent amounts of active enzyme present indifferent batches of the liver acetone powder.

SUMMARY OF THE INVENTION

[0007] Among the objects of the present invention, therefore, is theprovision of enzymatic processes for the resolution of enantiomericmixtures of β-lactams useful in the preparation of taxanes which offersimproved reproducibility as compared to processes which employ acetonepowders of animal livers and which compares favorably in cost toprocesses which employ purified lipases and other enzymes.

[0008] Briefly, therefore, the present invention is directed to aprocess for the resolution of a racemic mixture of β-lactams whichcontain an ester. The process comprises selectively hydrolyzing theester of one of the enantiomers by combining the mixture withhomogenized liver.

[0009] Other objects and features of this invention will be in partapparent and in part pointed out hereinafter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A. Starting Materials

[0011] In general, the β-lactam enantiomers in the mixture have thefollowing structural formula:

[0012] wherein

[0013] X₁ is —OX₆;

[0014] X₂ is hydrogen, hydrocarbyl, substituted hydrocarbyl, orheterocyclo;

[0015] X₃ is hydrogen, hydrocarbyl, substituted hydrocarbyl, orheterocyclo;

[0016] X₄ is hydrocarbyl, substituted hydrocarbyl, or heterocyclo;

[0017] X₅ is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX₁₀,—COOX₁₀, or —CONX₈X₁₀;

[0018] X₆ is acyl;

[0019] X₈ is hydrogen, hydrocarbyl, substituted hydrocarbyl, orheterocyclo; and

[0020] X₁₀ is hydrocarbyl, substituted hydrocarbyl, or heterocyclo.

[0021] Preferably, X₂ and X₃ are hydrogen and the mixture contains the3S,4R and 3R,4S enantiomers, and more preferably a racemic mixture ofthese enantiomers. Still more preferably, X₂ and X₃ are hydrogen; X₅ ishydrogen, hydrocarbyl, substituted hydrocarbyl, —COX₁₀, or —COOX₁₀; X₁₀is alkyl, aryl or heterocyclo; and the mixture contains the 3S,4R and3R,4S enantiomers, preferably a racemic mixture of these enantiomers. Ina particularly preferred embodiment, X₂ and X₃ are hydrogen; X₅ ishydrogen, hydrocarbyl, substituted hydrocarbyl, —COX₁₀, or —COOX₁₀; X₁₀is alkyl, aryl or heterocyclo; X₄ is 2-furyl, 3-furyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, substituted phenyl(substituted with any of the substituents identified elsewhere herein ashydrocarbyl substituents such as halo or nitro with the phenyl beingmono or poly substituted in one or more of the ortho, meta or parapositions), cycloalkyl, or alkenyl, and the mixture contains the 3S,4Rand 3R,4S enantiomers, preferably a racemic mixture of theseenantiomers.

[0022] Enantiomeric mixtures of the β-lactam starting materials may beobtained as described in Example 1 herein, or by methods analogous tothose described in U.S. Pat. No. 5,229,526 which is incorporated hereinby reference.

[0023] Homogenates of fresh or fresh frozen crude liver may be preparedusing a high speed blender or other grinder. The liver is ground intopieces of a relatively small size and suspended in an aqueous solution.Preferably, about 1 pound (about 450 grams) of liver is combined withsufficient liquid to form about 0.5 to about 2 liters of homogenate,more preferably about 0.75 to about 1.25 liters, and most preferablyabout 1 liter of homogenate. In a preferred embodiment, the homogenateis buffered, preferably to a pH of about 8 with a phosphate or othersuitable buffering agent.

[0024] Derivatives of homogenates are also included within theinvention, such as refined fractions. To obtain refined fractions onemay subject the homogenate to a series of fractionation procedures, thenumber of fractionation steps employed being dependent on the degree ofpurification desired. The series of fractionation steps could involvecolumn chromatography such as a gel filtration column, centrifugation,heat treatment, precipitation, filtration or various other appropriatemeans of purification.

[0025] In general, the liver may be obtained from any animal.Preferably, the liver is avian or mammalian, more preferably chicken,turkey, pig or beef, and most preferably beef.

[0026] Stereoselective Hydrolysis

[0027] In general, a solution of β-lactam mixture in an organic solventis combined with the homogenate to form a reaction mixture whichcontains about 1 gram of β-lactam to about 5 ml. to about 100 ml. ofhomogenate, more preferably about 1 gram of β-lactam to about 50 ml. ofhomogenate (with the homogenate containing about 450 grams of liver andsufficient liquid to form about 1 liter of homogenate). The reactionmixture is preferably adjusted to and maintained at about pH 7 to pH 8,preferably with a buffer, more preferably with a phosphate buffer.

[0028] The hydrolysis is preferably conducted in an aqueous, such as abuffered aqueous (e.g., phosphate buffer), medium or in an aqueousmedium containing a miscible or immiscible organic solvent. For example,the reaction may be conducted in a biphasic solvent system comprising anorganic phase, immiscible in water, and an aqueous phase.

[0029] Solvents for the organic phase of a biphasic solvent system maybe any organic solvent immiscible in water, such as toluene, benzene,hexane, cyclohexane, xylene, trichlorotrifluoroethane, dichloromethane,ether and the like, and is preferably ether or toluene. Typically, theconcentration of the β-lactam mixture will be about 0.1 to about 1millimolar. The aqueous phase is water, preferably deionized water, or asuitable aqueous buffer solution, especially a phosphate buffersolution. The biphasic solvent system preferably comprises between about10 to 90 percent by volume of organic phase and between about 90 to 10percent by volume of aqueous phase.

[0030] The reaction time may be selected based on the homogenate, thetemperature and the enzyme concentration. Temperatures of from about 4°C. to about 60° C. are preferably employed.

[0031] Separation

[0032] The products of the stereoselective conversions may be isolatedand purified by methodologies such as extraction, distillation,crystallization, column chromatography, and the like.

[0033] Definitions

[0034] The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Preferably, thesemoieties comprise 1 to 20 carbon atoms.

[0035] The “substituted hydrocarbyl” moieties described herein arehydrocarbyl moieties which are substituted with at least one atom otherthan carbon, including moieties in which a carbon chain atom issubstituted with a hetero atom such as nitrogen, oxygen, silicon,phosphorous, boron, sulfur, or a halogen atom. These substituentsinclude halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,hydroxy, protected hydroxy, keto, acyl, acyloxy; nitro, amino, amido,nitro, cyano, and thiol.

[0036] The alkyl groups described herein are preferably lower alkylcontaining from one to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includemethyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.

[0037] The alkenyl groups described herein are preferably lower alkenylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and thelike.

[0038] The alkynyl groups described herein are preferably lower alkynylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

[0039] The terms “aryl” or “ar” as used herein alone or as part ofanother group denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

[0040] The terms “halogen” or “halo” as used herein alone or as part ofanother group refer to chlorine, bromine, fluorine, and iodine.

[0041] The terms “heterocyclo” or “heterocyclic” as used herein alone oras part of another group denote optionally substituted, fully saturatedor unsaturated, monocyclic or bicyclic, aromatic or nonaromatichydrocarbon groups having at least one heteroatom in at least one ring,and preferably 5 or 6 atoms in each ring. The heterocyclo grouppreferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4nitrogen atoms in the ring, and may be bonded to the remainder of themolecule through a carbon or heteroatom. Exemplary heterocyclo includefuryl, thienyl, pyridyl and the like. Exemplary substituents include oneor more of the following groups: hydrocarbyl, substituted hydrocarbyl,keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy,alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, and thiol.

[0042] The acyl moieties described herein contain hydrocarbyl,substituted hydrocarbyl or heterocyclo moieties.

[0043] The term “stereoselective conversion,” as used herein, refers tothe preferential reaction of one enantiomer relative to another, thatis, asymmetric, enantioselective, reaction. Likewise, the terms“stereoselective hydrolysis”, refers to the preferential hydrolysis ofone enantiomer relative to another.

[0044] The term “mixture,” as said term is used herein in relation toenantiomeric compounds, denotes mixtures having equal (racemic) ornon-equal amounts of enantiomers.

[0045] The term “resolution” as used herein denotes partial, as well as,preferably, complete resolution.

[0046] The terms “hydroxyl protecting group” and “hydroxy protectinggroup” as used herein denote a group capable of protecting a freehydroxyl group (“protected hydroxyl”) which, subsequent to the reactionfor which protection is employed, may be removed without disturbing theremainder of the molecule. A variety of protecting groups for thehydroxyl group and the synthesis thereof may be found in “ProtectiveGroups in Organic Synthesis” by T. W. Greene, John Wiley and Sons, 1981,or Fieser & Fieser. Exemplary hydroxyl protecting groups includemethoxymethyl, l-ethoxyethyl, benzyloxymethyl,(.beta.-trimethylsilylethoxy)methyl, tetrahydropyranyl,2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl,trichloromethoxycarbonyl and 2,2,2-trichloroethoxymethyl.

EXAMPLES Example 1 Beef Liver Resolution of N-PMP-4-(2-furyl)-β-lactam

[0047]

[0048] To a 4-L Erlenmeyer flask equipped with a mechanical stirrer wasadded a solution of 62 g (0.205 mol) of recrystallized racemic β-lactamin 1.0 L of toluene. The solid was completely dissolved by warming witha 40° C. water bath and then cooling to ambient temperature. To a 1-Lgraduated cylinder was added 87.1 g (0.5 mol) of K₂HPO₄, and 4.9 g(0.036 mol) of KH₂PO₄ diluted to the 1000 mL mark with RO water andmixed to dissolve completely. A beef liver suspension was prepared byadding 20 g of frozen beef liver (Premium Select Brand, Nebraskaland,Inc., New York) to a blender and then adding the pH 8 buffer to make atotal volume of 1 L. The mixture was blended to make a homogeneoussuspension which was then added directly to the toluene solution. Themixture was stirred for 22 h at ambient temperature. The layers wereseparated and the aqueous layer was extracted with 1 L of ethyl acetate.The organic layers were combined and concentrated to give 67 g of whitepowder. The powder was recrystallized from 600 mL of hot absoluteethanol to give 23.4 g (0.078 mol, 38%) of optically pure β-lactam(+)-m.p. 153-155° C.; [a]²⁰ ₅₈₉=+38.1° (MeOH, c=0.7).

Example 2

[0049] As noted previously, others have reported successful applicationof pig liver acetone powder (PLAP), bovine liver acetone powder (BLAP)and chicken liver acetone powder (CLAP), in the resolution of numerouschiral secondary alcohols. The application of a BLAP biphasic enzymaticresolution procedure to (±)-1a resulted in 37% yield of the hydroxyβ-lactam (−)-2a and a 33% yield of the acetoxy β-lactam (−)-1a after 3 hat room temperature (Scheme 1).

[0050] The hydroxy β-lactam (−)-2a was shown to be optically pure bycomparison to the reported specific rotation and by ¹H NMR studies onits Mosher ester as well as Eu(hfc)₃ chemical shift analysis on thealcohol. The optical purity of recovered acetate (−)-1a varied from76%ee to >95%ee, and was found to be dependent upon the batch of liveracetone powder used. Conversion of acetate (−)-1a to the desired hydroxyβ-lactam (+)-2a was carried out using Liu's pyrolidine-pyridineprocedure. Liu, J. H., Ph.D. Dissertation, The Florida State University,1991. Thus, liver acetone powder is highly selective toward the 3Senantiomer of the β-lactam. This selectivity was further confirmed bythe fact that the optically pure (−)-3R,4S-β-lactam 1a was nothydrolyzed under the same conditions after 24 h. Therefore, the lowoptical purity of the recovered (−)-1a is the result of incompletereaction rather than the enzyme's lack of selectivity. This is probablya consequence of inconsistent amounts of active enzyme present indifferent batches of the liver acetone powder. It was reasoned that thisinconsistency could be eliminated if the enzyme was not subjected to thedrying process in the acetone powder preparation.

[0051] Hence, a phosphate buffered beef liver solution (BBLS) wasprepared by homogenizing 1 lb of frozen beef liver in a 0.5 MKH₂PO₄/K₂HPO₄ pH 8 buffer to make 1 L of total solution. A 50 mL aliquotof this BBLS was found to selectively hydrolyze the (+)-1a isomer from1.0 g of (±)-1a in 15 minutes, leaving the (−)-1a isomer unchanged. Theenantiomeric excess of both the acetate and the alcohol was found to beessentially 100% as determined by the methods described previously.

[0052] Similarly, buffered chicken (BCLS), pig (BPLS), and turkey (BTLS)liver solutions showed the same activity toward (±)-1a. Table Isummarizes the enzyme sources used in the resolution and theirefficiencies. TABLE I Efficacies of Various Enzyme Sources in theResolution of (±)-1a (−)-1a (−)-2a Enzyme ^(α)578²⁵ ^(α)578²⁵ TimeSource Yield (%) CHCL₃ % ee Yield (%) CHCL₃ % ee (hrs) BLAP 33 −40.0 7837 −178.6 >95 3.0 BBLS 48 −46.9 >95 45 −178.7 >95 0.25 BCLS 53 −40.0 7845 −175.6 >95 0.25 BTLS 55 −40.5 78 31 −179.4 >95 0.25 BPLS 62 −30.8 6536 −175.8 >95 0.25

[0053] It is unclear which enzyme in the liver is responsible for thisenantioselective lipase activity. Undoubtedly, there is more than oneactive enzyme in a crude solution of beef liver. Nevertheless, thisenzymatic methodology was found to be ideally suited for the preparationof other optically active β-lactam analogs. Table II shows a variety ofβ-lactam substrates which were resolved by BBLS. As indicated in thetable, the BBLS enzymatic resolution procedure proved effective foralmost all of the substrates examined. Problems did arise in thehydrolysis of substrates containing highly polar groups. For example,when R⁴=p-nitrophenyl and R^(N)=H, the resulting alcohol was notisolable. Similar problems were also encountered with substrates whereR⁴=pyridyl. It is unclear whether the product was lost to the aqueouslayer in these reactions or underwent decomposition during the course ofthe reaction.

[0054] To remedy this problem, the R^(N) group was changed to the morehydrophobic p-methoxyphenyl (PMP) group. As can be seen from Table II,introduction of the PMP group increased the time required for thecomplete hydrolysis of the 3S,4R enantiomer. This is probably due to theloss of a hydrogen bonding site in addition to the increase in stericbulk. Nonetheless, this allowed the alcohol product to be isolated andcharacterized. Interestingly, it was found that the hydrolyzed productsfrom the substrates having R⁴=2-pyridyl, 4-pyridyl and 4-nitrophenylwere a mixture of cis and trans hydroxy β-lactams.

[0055] Currently, this enzymatic resolution procedure is the mostgeneral and convenient protocol for the production of various opticallyactive β-lactams suitable for the preparation of taxol C13 side chainanalogs. TABLE II BBLS Resolution of Various β-lactams (±)-1a-o AcetateAlcohol Time yield ^(α)578²⁵ yield ^(α)578²⁵ 1 R⁴ R^(N) (h) (%) CHCL₃ %ee (%) CHCL₃ % ee a Phenyl H 0.25 46 −46 >95 45 −175 >95 b 2-Thienyl H0.25 42 −93 >95 49 −135 >95 c 3-Thienyl H 2 49 −82 >95 25 −119 >95 d2-furyl H 0.25 40 −125 >95 40 −107 >95 e 3-furyl H 1.2 43 −91 >95 38−86 >95 f p-Bromo-phenyl H 0.25 54 −36 71 39 −122 >95 g1-(2-methyl-1-propenyl) H 4 45 −23 >95 33 −100 >95 h Cyclobutyl H 1 40−79 >95 49 −29 >95 i p-Nitrophenyl H 1 38 −49 90 0 — — j 2-Pyridyl H 0.545 −5 >95 0 — — k Phenyl PMP 24 42 +8 >95 45 −172 >95 l 2-Pyridyl PMP 1642 +42 >95 30 Mixture of m 3-Pyridyl PMP 10 33 +8.7 >95 45 cis + trans n4-Pyridyl PMP 10 55 +25 75 43 o p-Nitrophenyl PMP 24 35 +48 >95 14

[0056] Materials and Methods

[0057] Frozen livers were bought from a local grocery store. Beef liverwas packed by Fremont Beef Company, Fremont, Nebr. 68025.

[0058] General Procedure for Preparation of Liver Acetone Powder. BeefLiver Acetone Powder (BLAP). Frozen beef liver (250 g) was homogenizedwith a blender, and 200 mL of acetone was added. The precipitate wascollected by filtration through coarse filter paper and dried under highvacuum for 2 h. The dried mass was further powderized with a blender togive 78 g BLAP and was stored at −30° C.

[0059] General Procedure for Liver Acetone Powder Mediated Hydrolysis.P-Lactam (−)-1a. To a solution of 1.0 g (4.87 mmol) of (±)-1a in 10 mLof diethyl ether at 25° C. was added 40 mL of 0.5 M phosphate buffer and1.0 g of BLAP. After 3 h, 100 mL of brine was added. After 10 min, themixture was diluted with 200 mL of ethyl acetate. The organic layer wasseparated, washed with brine and dried over Na₂SO₄ and concentrated.Flash chromatography eluting with 75% ethyl acetate in hexanes gave 0.33g (1.6 mmol) of (−)-1a as the less polar fraction and 0.29 g (1.8 mmol)of (−)-2a as the polar fraction.

[0060] General Procedure for Phosphate Buffer Liver Solution. BufferedBeef Liver Solution (BBLS). Frozen beef liver (500 g) was homogenizedwith a blender. The mixture was diluted to 1 L of volume with pH 8 (˜0.5M of PO₄ ⁻²) phosphate buffer and stored at −30° C. (no loss of enzymeactivity after 15 days).

[0061] General Procedure for Buffered Liver Solution MediatedHydrolysis. β-Lactam (−)-1a. To a solution of 1.0 g (4.87 mmol) of(±)-1a in 100 mL of diethyl ether at 25° C. was added 50 mL of the BBLSand 50 mL of phosphate buffer (0.5 M, pH 8). After 15 min, 10 mL ofbrine was added. The reaction mixture was diluted with 200 mL ofacetone, filtered and concentrated. The residue was diluted with 200 mLof ethyl acetate, washed with brine, dried over Na₂SO₄ and concentratedto give a yellow solid. Flash chromatography eluting with 75% ethylacetate in hexane gave 0.480 g (2.34 mmol, 48%) of (−)-1a as the lesspolar fraction and 0.354 g (2.17 mmol, 45%) of (−)-2a as the polarfraction.

[0062] 1a: mp 187-189° C.; [α]₅₇₈ ²⁵=−46° (c=1.0, CHCl₃); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.67 (s, 3H), 5.04 (d, J=4.5 Hz, 1H), 5.89(dd, J=4.5, 2.8 Hz, 1H), 6.22 (bm, 1H), 7.34 (m, 5H).

[0063] 2a: mp 190-192° C.; [α]₅₇₈ ²⁵=−175° (c=1.0, MeOH); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.95 (d, J=9.9 Hz, 1H), 4.96 (d, J=4.6 Hz,1H), 5.12 (m. 1H), 6.15 (bm, 1H), 7.41 (m, 5H);

[0064] β-Lactam (−)-1b. Following the general procedure, 0.57 g (0.65mmol) of (±)-1b was treated with BBLS for 0.25 h to give 0.225 g (0.14mmol, 30%) (−)-1b.

[0065] (−)-1b: mp 135-136° C.; [α]₅₇₈ ²⁵=−93° (c=1.0, CHCl₃); ¹H NMR(300 MHZ, CDCl₃) δ (ppm): 1.84 (s, 3H), 5.28 (d, J=4.6 Hz, 1H), 5.87(dd, J=4.6, 2.7 Hz, 1H), 6.55 (bm, 1H), 7.02 (m, 2H), 7.33 (dd, J=4.6,1.7 Hz, 1H).

[0066] 2b: mp 144-145° C.; [α]₅₇₈ ²⁵=−135° (c=1.0, MeOH); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 2.55 (d, J=9.7 Hz, 1H), 5.1 (m, 1H), 5.17(d, J=5.0 Hz, 1H), 6.33 (bm, 1H), 7.10 (m, 2H), 7.37 (dd, 4.5, 1.3 Hz,1H).

[0067] β-Lactam (−)-1d. Following the general procedure, 0.57 g (2.92mmol) of (±)-1d was treated with BBLS for 0.25 h to give 0.225 g (1.15mmol, 40%) of (−)-1d and 0.16 g (1.04 mmol, 35%) of (−)-2d.

[0068] (−)-1d: mp 158-159° C.; [α]₅₇₈ ²⁵=−134° (c=1.0, CHCl₃); >95% ee;¹H NMR (300 MHZ, CDCl₃) δ (ppm): 1.90 (s, 3H), 5.04 (d, J=5.0 Hz, 1H),5.89 (d, J=5.0, 2.0 Hz, 1H), 6.20 (bm, 1H), 6.40 (bm, 2H), 7.44 (s,1H)

[0069] 2d: mp 144-145° C.; [α]₅₇₈ ²⁵=−107° (c=1.0, MeOH); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 2.78 (bm,1H), 3.49 (d, J=5.0 Hz, 1H), 4.91(d, J=5.0 Hz, 1H), 5.12 (bm, 1H), 6.13 (bm, 1H), 6.45 (m, 2H), 7.49 (d,J=1.2 Hz, 1H)

[0070] β-Lactam (−)-1i. Following the general procedure, 0.1 g (0.4mmol) of (±)-1i was treated with BBLS for 1 h to give 0.030 g (0.14mmol, 30%) (−)-1i.

[0071] (−)-1i: mp 198-199° C.; [α]₅₇₈ ²⁵=−49° (c=1.0, CHCl₃); 90% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.73 (s, 3H), 5.16 (d, J=4.7 Hz, 1H), 5.95(dd, J=4.7, 3.1 Hz, 1H), 6.58 (bm, 1H), 7.52 (d, J=8.5 Hz, 2H), 8.24 (d,J=8.5 Hz, 2H)

[0072] β-Lactam (−)-1j. Following the general procedure, 1.0 g of (±)-1jwas treated with BBLS for 24 h to give 0.223 g of (−)-1j.

[0073] (−)-1j: mp 99-100° C.; [α]₅₇₈ ²⁵=−5° (c=1.0, CHCl₃); >95%ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.74 (s, 3H), 5.15 (d, J=5.1 Hz, 1H), 6.06(dd, J=5.1, 2.3 Hz, 1H), 6.46 (bm, 1H), 7.26 (m, 1H), 7.36 (d, J=7.8 Hz,1H), 7.73 (ddd, J=8.5, 7.4, 2.1 Hz, 1H), 8.61 (d, J=4.9 Hz, 1H).β-Lactam (−)-1k. Following the general procedure, 1.0 g (3.21 mmol) of(±)-1k was treated with BBLS for 24 h to give 0.42 g (1.35 mmol, 42%) of(+)-1k and 0.377 g (01.44 mmol, 45%) of (−)-2k.

[0074] (+)-1k: mp 164-165° C.; [α]₅₇₈ ²⁵=+8° (c=1.0, CHCl₃); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.67 (s, 3H), 3.75 (s, 3H), 5.34 (d, J=5.0Hz, 1H), 5.94 (d, J=5.0 Hz, 1H), 6.81 (d, J=8.4 Hz, 2H), 7.31 (m, 7H)

[0075] β-Lactam 1l: mp 154-155° C.; [α]₅₇₈ ²⁵=+42° (c=1.0, CHCl₃); >95%ee; ¹H NMR (300 MHZ, CDCl₃) δ (ppm): 1.74 (s, 3H), 3.76 (s, 3H), 5.48(d, J=5.3 Hz, 1H), 6.12 (d, J=5.3 Hz, 1H), 6.81 (d, J=9.1 Hz, 2H), 7.29(m, 4H), 7.68 (ddd, J=7.7, 7.8, 1.7, Hz, 1H), 8.64 (d, J=4.7 Hz, 1H);

[0076] β-Lactam 1m: mp 158-160° C.; [α]₅₇₈ ²⁵=+8.7° (c=1.0, CHCl₃); >95%ee; ¹H NMR (300 MHZ, CDCl₃) δ (ppm): 1.74 (s, 3H), 3.76 (s,3H), 5.38 (d,J=4.5 Hz, 1H), 5.98 (d, J=4.5 Hz, 1H), 6.81 (d, J=9.1 Hz, 2H), 7.25 (d,J=9.1 Hz, 2H), 7.30 (m, 1H), 7.64 (m, 1H), 8.61 (m, 2H).

[0077] β-Lactam 1n: mp 158-159° C.; [α]₅₇₈ ²⁵=+25° (c=1.0, CHCl₃); >95%ee; ¹H NMR (300 MHZ, CDCl₃) δ (ppm): 1.74 (s, 3H), 3.76 (s, 3H), 5.32(d, J=5.0 Hz, 1H), 5.98 (d, J=5.0 Hz, 1H), 6.83 (d, J=9.1 Hz, 2H), 7.23(m,4H), 8.62 (m, 2H);

[0078] β-Lactam 2n-trans: ¹H NMR (300 MHZ, CDCl₃) δ (ppm): 3.75 (s, 3H),4.70 (s, 1H), 4.84 (s, 1H), 6.79 (m, 2H), 7.16 (m, 2H), 7.24 (d, J=5.5Hz, 2H), 8.61 (d, J=5.5 Hz, 2H).

[0079] β-Lactam 1o. Following the general procedure, 1.0 g (2.8 mmol)was treated with BBLS for 24 h to give 0.35 g (1.11 mmol, 35%) of(+)-1o, 0.11 g (0.35 mmol, 12%) of 2o and 0.13 g (0.41 mmol, 15%) of2o-trans.

[0080] 1o: mp 156-160° C.; [α]₅₇₈ ²⁵=+48° (c=1.0, CHCl₃); >95% ee; ¹HNMR (300 MHZ, CDCl₃) δ (ppm): 1.74 (s, 3H), 3.76 (s, 3H), 5.45 (d, J=5.1Hz, 1H), 6.00 (d, J=5.1 Hz, 1H),6.83 (d, J=8.9 Hz, 2H), 7.23 (d, J=8.9Hz, 2H), 7.50 (d, J=9.2 Hz, 2H), 8.23 (d, J=9.2 Hz, 2H);

[0081] 2o: mp 158-159° C.; [α]₅₇₈ ²⁵=−52° (c=1.0, MeOH); >95% ee; ¹H NMR(500 MHZ, CDCl₃) δ (ppm): 2.37 (m, 1H), 3.76 (s, 3H), 5.31 (m, 1H), 5.35(d, J=5.0 Hz, 1H), 6.83 (d, J=9.1 Hz, 2H), 7.24 (d, J=9.1 Hz, 2H), 7.52(d, J=8.8, 2H), 8.27 (d, J=8.8, 2H);

[0082] 2o-trans: mp 98-100° C.; ¹H NMR (500 MHZ, CDCl₃) δ (ppm): 3.40(d, J=6.2 Hz, 1H), 3.74 (s, 3H), 4.73 (dd, J=6.3, 1.1 Hz, 1H), 6.79 (d,J=9.3 Hz, 2H), 7.16 (d, J=9.3 Hz, 2H), 7.51 (d, J=9.3 Hz, 2H), 8.25 (d,J=9.3 Hz, 2H).

What is claimed is:
 1. A process for the resolution of an enantiomericmixture of an acyloxy substituted β-lactam, the process comprising (a)homogenizing a liver; (b) fractionating the homogenized liver to form apartially purified liver homogenate; (c) combining the enantiomericmixture with the partially purified liver homogenate to form a reactionmixture; (d) selectively hydrolyzing the acyloxy substituent of one ofthe enantiomers in the reaction mixture; and (e) recovering a β-lactamhaving an unhydrolyzed acyloxy substituent from the reaction mixture. 2.The process of claim 1 wherein the enantiomeric mixture is a racemicmixture.
 3. The process of claim 1 wherein the enantiomeric mixture is amixture of the 3S, 4R and the 3R,4S enantiomers.
 4. The process of claim3 wherein the enantiomeric mixture is a racemic mixture.
 5. The processof claim 1 wherein the partially purified liver homogenate is partiallypurified avian liver homogenate or partially purified mammalian liverhomogenate.
 6. The process of claim 1 wherein the partially purifiedliver homogenate is a partially purified beef liver homogenate.
 7. Theprocess of claim 1 wherein the enantiomeric mixture comprises anethereal solvent.
 8. The process of claim 1 wherein the enantiomericmixture comprises toluene solvent.
 9. The process of claim 1 wherein theβ-lactam has the formula:

wherein X₁ is —OX₆; X₂ is hydrogen, hydrocarbyl, substitutedhydrocarbyl, or heterocyclo; X₃ is hydrogen, hydrocarbyl, substitutedhydrocarbyl, or heterocyclo; X₄ is hydrocarbyl, substituted hydrocarbyl,or heterocyclo; X₅ is hydrogen, hydrocarbyl, substituted hydrocarbyl,—COX₁₀, —COOX₁₀, or —CONX₈X₁₀; X₆ is acyl; X₈ is hydrogen, hydrocarbyl,substituted hydrocarbyl, or heterocyclo; and X₁₀ is hydrocarbyl,substituted hydrocarbyl, or heterocyclo.
 10. The process of claim 9wherein X₂ and X₃ are hydrogen.
 11. The process of claim 9 wherein X₂and X₃ are hydrogen, X₅ is hydrogen, hydrocarbyl, substitutedhydrocarbyl, —COX₁₀, or —COOX₁₀, and X₁₀ is as defined in claim
 9. 12.The process of claim 9 wherein X₅ is hydrogen, hydrocarbyl, substitutedhydrocarbyl, —COX₁₀, or —COOX₁₀, and X₁₀ is as defined in claim
 9. 13.The process of claim 9 wherein X₂ is hydrogen, X₃ is hydrogen, and X₄ isheterocyclo.
 14. The process of claim 9 wherein X₂ is hydrogen, X₃ ishydrogen and X₄ is furyl.
 15. The process of claim 14 wherein thepartially purified liver homogenate is partially purified avian liverhomogenate or partially purified mammalian liver homogenate.
 16. Theprocess of claim 9 wherein the partially purified liver homogenate ispartially purified avian liver homogenate or partially purifiedmammalian liver homogenate.
 17. The process of claim 9 wherein thepartially purified liver homogenate is partially purified beef liverhomogenate.
 18. The process of claim 17 wherein X₄ is heterocyclo. 19.The process of claim 9 wherein X₂ and X₃ are hydrogen and theenantiomeric mixture contains the 3S,4R and 3R,4S enantiomers.
 20. Theprocess of claim 19 wherein the enantiomeric mixture is a racemicmixture of the 3S,4R and 3R,4S enantiomers.
 21. The process of claim 20wherein the partially purified liver homogenate is partially purifiedavian liver homogenate or partially purified mammalian liver homogenate.22. The process of claim 20 wherein the partially purified liverhomogenate is partially purified beef liver homogenate.
 23. The processof claim 9 wherein X₂ and X₃ are hydrogen, X₅ is hydrogen, hydrocarbyl,substituted hydrocarbyl, —COX₁₀, or —COOX₁₀, X₁₀ is alkyl, aryl orheterocyclo and the mixture contains the 3S,4R and 3R,4S enantiomers.24. The process of claim 23 wherein the enantiomeric mixture is aracemic mixture of the 3S,4R and 3R,4S enantiomers.
 25. The process ofclaim 24 wherein the partially purified liver homogenate is partiallypurified avian liver homogenate or partially purified mammalian liverhomogenate.
 26. The process of claim 24 wherein the partially purifiedliver homogenate is partially purified beef liver homogenate.
 27. Theprocess of claim 9 wherein X₂ and X₃ are hydrogen, X₅ is hydrogen,hydrocarbyl, substituted hydrocarbyl, —COX₁₀, or —COOX₁₀; X₁₀ is alkyl,aryl or heterocyclo; X₄ is 2-furyl, 3-furyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-thienyl, 3-thienyl, substituted phenyl, cycloalkyl, oralkenyl and the mixture contains the 3S,4R and 3R,4S enantiomers. 28.The process of claim 27 wherein the enantiomeric mixture is a racemicmixture of the 3S,4R and 3R,4S enantiomers.
 29. The process of claim 28wherein the partially purified liver homogenate is partially purifiedavian liver homogenate or partially purified mammalian liver homogenate.30. The process of claim 28 wherein the partially purified liverhomogenate is partially purified beef liver homogenate.
 31. The processof claim 9 wherein X₂ and X₃ are hydrogen and the reaction mixturecomprises the 3S,4R and 3R,4S enantiomers and partially purified liverhomogenate.
 32. The process of claim 9 wherein X₂ and X₃ are hydrogen,X₅ is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX₁₀, or—COOX₁₀, X₁₀ is alkyl, aryl or heterocyclo and the reaction mixturecomprises the 3S,4R and 3R,4S enantiomers and partially purified liverhomogenate.
 33. The process of claim 32 wherein the partially purifiedliver homogenate is partially purified beef liver homogenate.
 34. Theprocess of claim 9 wherein X₂ and X₃ are hydrogen, X₅ is hydrogen,hydrocarbyl, substituted hydrocarbyl, —COX₁₀, or —COOX₁₀; X₁₀ is alkyl,aryl or heterocyclo; X₄ is 2-furyl, 3-furyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-thienyl, 3-thienyl, substituted phenyl, cycloalkyl, oralkenyl and the reaction mixture comprises the 3S,4R and 3R,4Senantiomers and partially purified liver homogenate.
 35. The process ofclaim 34 wherein the partially purified liver homogenate is partiallypurified beef liver homogenate.